Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from two German hospitals

Introduction: Vancomycin-resistant Enterococcus faecium accounts for around 10–23% of nosocomial enterococcal infections and constitutes a relevant therapeutic problem due to its limited susceptibility to antibiotics. The resistance towards glycopeptide antibiotics is mediated by the so-called van genes. Currently, the most common resistance type in Germany is the vanB-type. Little data are available on the molecular epidemiology in Germany. Therefore, an epidemiological typing of Enterococcus faecium isolates with vanB-type resistance from two German hospitals in Essen and Nuremberg was performed. Two outbreaks and 104 sporadic cases were investigated. Methods: All 128 isolates with vanB-type resistance were collected between 2011–2012 and 2017–2018. They were characterized using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Results: ST 117 was the most common sequence type (ST) in both hospitals, especially since 2017. PFGE divided the isolates of this study into 68 PFGE types and showed a broad genetic diversity. Two epidemiologically assumed in-hospital outbreaks were genetically confirmed. Apart from that, in-hospital transmissions were rare events. Conclusion: The results obtained by MLST confirmed the previously described allocation of STs in Germany. PFGE showed a broad genetic diversity of vanB VRE between the two hospitals and also within each hospital. In-hospital transmissions were rare, but outbreaks did occur. Our data supports the strategy to screen and isolate patients in transmission events in order to detect monoclonality indicating a common source or hygiene mismanagement.


Introduction
Enterococci are gram-positive, catalase-negative and facultative anaerobic opportunistic bacteria that can be found as a part of the gastrointestinal microbiota of humans [1], [2]. They can be especially relevant, or pathogenic, for immunocompromised patients, causing different infections [3]. In 2016, a national point prevalence survey reported that 12.6% of the included nosocomial infections were caused by enterococci [4]. Similar to other countries, an increasing rate of resistance to vancomycin (VRE) has been reported for Germany [4]. The most clinically relevant Enterococcus species is Enterococcus faecium (E. faecium) [2], [5], being the most frequently resistant species against glycopeptides [6], which also causesmost of the detected enterococcal nosocomial infections [7]. The most common resistance type of E. faecium in Germany is vanB (phenotypic resistance to vancomycin only) followed by vanA (phenotypic resistance to vancomycin and teicoplanin) [8]. In this study, the molecular epidemiology of invasive VRE isolates with the vanB phenotype from two German hospitals was analysed. The isolates were characterised by two molecular typing methods to investigate previous outbreaks, and understand possible transmission origins as well as further transmission routes.

Vancomycin resistant enterococcal isolates
Of 146 samples, 128 isolates were vanB-positive and belonged to the species E. faecium, meeting the inclusion criteria for this study. The 128 clinical isolates were collected in the years 2011-2013 and 2017-2018 at the University Hospital Essen (N=81) and in 2018 at the Hospital Nuremberg (N=47), Germany. In brief, 41% of the isolates were retrieved from patients in intensive care units in both hospitals, 8% were derived from the perinatal centre (outbreak cluster) of the University Hospital Essen and the remaining 51% from units of the other various departments. The isolates were derived from different clinical material. The majority were isolated from rectal swabs, blood cultures (N=20) and urine. No duplicate isolates from a single patient were included. Isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). They were conserved in cryotubes at -20°C and cultivated on Columbia agar with sheep blood (Oxoid, Wesel, Germany) overnight at 36°C before DNA extraction.

DNA extraction
For DNA extraction, the Maxwell16 (Promega, Madison, Wisconsin, USA) was used according to the manufacturer's instructions [9].

PCR determination of species and glycopeptide resistance genotype
Species and resistance genes were determined using a specific PCR, as previously described by Dutka-Malen et al. [10]. Isolates that were not found to be vanB-positive E. faecium were excluded.

Multilocus sequence typing (MLST)
MLST was carried out with a set of primers amplifying the seven housekeeping genes adk, atpA, ddl, gyd, gdh, purK, and pstS, according to procedures described by Homan et al. [11]. The obtained sequences were then downloaded from the web page of the LGC genomics shop (https://shop.lgcgenomics.com/) and inserted into the MLST database (https://pubmlst.org/bigsdb?db=pubmlst_efaecium_isolates) to determine the sequence types (ST) of the isolates. A phylogenetic analysis was performed with PHYLOViZ software (http://www.phyloviz.net/) using the goeBURST algorithm.

Pulsed-field gel electrophoresis (PFGE)
All isolates were characterised by PFGE using the technology from Bio-Rad Laboratories (Hercules, California, USA), based on protocols by Miranda et al. [12]. Briefly, the bacteria were embedded in agarose plugs before the cell walls were lysed and the bacterial genome was digested with the restriction enzyme SmaI. The resulting fragments were separated in a 1% agarose gel applying an alternating electric field with 6 V of electric tension at an angle of 120°. The pulse times were 5 s for 4 h, 10 s for 10 h and 25 s for 10 h. The DNA patterns in the gels were stained with ethidium bromide and photo documented with a BioVision device (Vilter, Cudahy, USA). The banding patterns were analysed with CLIQS 1D Pro 1.3.063 software from TotalLab (settings: Jaccard and WPMGA) and visual inspection according to the criteria described by Tenover et al. for outbreaks of Staphylococcus aureus [13].

Results
From a total of 146 initially analysed enterococcal isolates, 18 were rejected after PCR because they did not meet the inclusion criteria (E. faecium and vanB-positive). Overall, 128 isolates remained for characterisation with MLST and PFGE. In MLST, the isolates could be assigned to one of a total of 15 detected STs. For some combinations of the seven alleles, no ST was deposited in the database, and when only 6 alleles were included, there was a variety of possible STs. To nevertheless include these isolates in the analysis, they were named after a one-variant ST and marked with an apostrophe (e.g., ST 117'). Furthermore, five isolates were assigned to two different STs, because in a few cases, the two alleles for one gene differed from each other. If both allele combinations were present in the database, the isolates were named after both (e.g., ST  After cluster analysis with the goeBURST algorithm (PHYLOViZ software), the distribution of STs was depicted in a tree diagram (Figure 1). The isolates cluster in a large group with two sub-groups around the two initial isolates ST 117 and ST 192, only differing from these in not more than two of the seven housekeeping genes. In this group, a distinction can be made between one MLST pool from Essen and one from Nuremberg (  ST 17',ST 80'). After analysis of the PFGE gels, a dendrogram indicating genetic relationship was generated (Figure 2). The cut-off line is at 90%, separating the 128 isolates into 68 PFGE types that differ in no more than one band. The largest fraction of the isolates (N=15, 12%) belongs to PFGE type 2. All 15 isolates were found on cardiological units of the University Hospital Essen between May and July 2011, with twelve of them on the two intensive care units. Overall, 14 isolates showed a banding pattern that was designated PFGE type 38. Three of those isolates originated from Essen between June and July 2018, and the remaining eleven isolates from various units of the Nuremberg hospital between August and December 2018. With ten isolates showing that banding pattern, PFGE type 33 was identified as the third most common PFGE type. Nine of the ten isolates were found in December 2017 on the intensive care unit and the general neonatology ward of the University Hospital Essen in rectal swabs. The tenth isolate was from the West German Tumour Centre in Essen, where it was also found in a rectal swab seven months later. In most of the cases, a differentiation between PFGE types from Essen and Nuremberg has been detected. Only five of the 68 PFGE types were found in both hospitals: PFGE type 34, 38, 40, 42 and 44. All the other PFGE types were limited to one facility. When comparing the classification of the isolates into STs by MLST and PFGE types, they mostly agree with each other. Most isolates of one ST could be divided into different PFGE types, with the following exceptions. Some PFGE types contained isolates with different STs. 14     The scale shows the similarity between the isolates. The shorter the distance, the greater the degree of relationship. The branches of the isolates that split on the right side of the grey line at 90% belong to one PFGE type (group).

Discussion
The 128 vanB-positive E. faecium isolates from Essen and Nuremberg were divided into 15 STs by MLST analysis. After phylogenetic analysis with the goeBURST algorithm, the two initial ST 117 and 78 could be determined. The initial isolates probably had a survival advantage in the particular environment, and therefore they could easily disperse and proliferate [14]. With time, some of the seven housekeeping genes changed through mutation, which led to new STs that still belonged to the same group [14]. The isolates from Essen and Nuremberg differ for the most part in their STs, which leads to the assumption that the isolates developed and adapted differently due to their different geographical origin. The 15 STs found in this study all belong to a clonal complex 17 (CC 17) [14], a group of VRE isolates that is responsible for nosocomial infections worldwide [15]. This subpopulation has been shown to be particularly pathogenic [16]. CC 17 is characterised by resistance to ampicillin [15], the purK allele 1 [17] and a pathogenicity island linked to the esp virulence gene [18]. In this study, 43 isolates derived from the hospital in Essen between 2011 and 2012, with 32 of these belonging to ST 192. This confirms what the Robert Koch Institute (RKI) described for this time period, with ST 192 being the most prevalent ST detected in Germany with 33% [19]. ST 117 was the most common ST overall in this study, both in Essen and Nuremberg and especially currently. This also is in line with the situation shown by the RKI in the last few years: in 2015 and 2016, ST 117 was the most frequently detected ST of the received isolates, followed by ST 203 and ST 80 [7]. In this study, ST 80 was overall the third-most and in Nuremberg the second-most frequently found ST. In a recently published study, ST 80 and ST 117 were the most common clonal groups in eight hospitals in Bavaria, Germany [20]. The 15 isolates belonging to PFGE type 2 and nine of ten isolates of PFGE type 33 were found in a short period of time and in a small area in the Essen hospital. This confirms the two VRE outbreaks in the cardiology department in 2011 and in the perinatal centre in 2017. In both outbreaks, most isolates only colonised patients' intestines. There were three bloodstream infections in the 2011 outbreak and one shunt-meningitis in the 2017 outbreak. Both outbreaks were terminated by extensive hygienic measures, such as patient screening, isolation, intensive surface cleaning and closing of the ward (personal communication, Peter-Michael Rath). The tenth isolate of PFGE type 33 was found seven months later in the West German Tumour Centre, which is spatially separate from the outbreak ward. However, complex transmission paths of VRE between different units were previously described by Raven et al. [21]. Some PFGE types were found in both Essen and Nuremberg. It might be possible that a clone spread over this distance. Other studies already described such cross-regional transmissions of PFGE types [1], [7]. However, due In conclusion, MLST separated the isolates into a few closely related STs, whereas PFGE showed a broad genetic diversity not only between the two hospitals in Essen and Nuremberg but also within them. Two outbreaks in the hospital in Essen were already epidemiologically recognised by the sudden appearance of VRE in clinical material of numerous patients. Apart from these outbreaks, transmission of VRE within the hospital occur more rarely than assumed. It is more likely that many of the patients acquire their own strain of VRE outside the hospital and then carry it along when hospitalised. Given the well-known low pathogenicity of enterococci for most patient groups (except hemato-oncological patients), our data support the strategy of screening and isolating patients only in outbreaks in order to detect monoclonality which indicates hygienic failures.

Competing interests
The authors declare that they have no competing interests.

Compliance with Ethical Standards
Isolates were analysed anonymously in a retrospective manner. Ethical approval and informed consent were not required.

Availability of data and material
The datasets generated and analysed during the current study are either included in this published article or available from the corresponding author on request.